1. Field of the Invention
The present invention relates to a method for the expression of biotin using edible yeast as a host, and more particularly, to a method for highly expressing biotin using edible yeast transformed by a genetically engineered plasmid.
2. Description of the Related Arts
Biotin (2′-keto-3,4-imidazolido-2-tetrahydrothiophene-n-valeric acid) was isolated from egg yolk by Kogl and Tonuis in 1936 and named as vitamin H. It is one of the essential vitamins for nutrition of animals, plants, and microorganisms, and very important as medicine or as a food additive. In general, biotin serves as a CO2 carrier for covalent binding in cells, and plays the role of coenzyme for carboxylase, decarboxylase, and transcarboxylase.
Biotin biosynthesis of Escherichia coli has been studied well, and the related genes are clarified. The structures and regulations of the bio operon in E. coli also have been thoroughly identified (Shiuan, D., et al., Gene 145:1–7, (1994)). This operator is located at the 17 min on the E. coli genomic map, which includes 5 key-enzyme genes for the biotin biosynthesis (referring to FIG. 1). The transcription of the 5 genes of the bioABFCD operon is controlled by a regulatory region located between bioA and bioB. This regulatory region consists of a 40-bp long operator sequence, partially overlapping the promoter. In addition, bioA gene and bioBFCD genes are transcribed bi-directionally from the regulatory region.
Another birA gene whose product is the repressor of the operator is located at the 89 min on the E. coli genomic map. The repression of transcription of the operator requires the actions of the repressor and co-repressor (i.e. biotinyl-5′-adenylate) together with the operator. Further, the biotinyl-5′-adenylate is produced by the activation of biotin by the gene product of the birA (one function of which is bio repressor, and another is biotin holo enzyme synthetase).
With respect to yeast, it is known that there are 3 biosynthesis genes in wine-making yeast, Saccharomyces cerevisae: BIO2 (encoding for biotin synthase), BIO3 (encoding for 7,8-diaminopelargonic acid aminotransferase), and BIO4 (encoding for dethiobiotin synthase), which correspond to bioB, bioA, and bioD genes in E. coli, respectively. That is, the last three steps of biotin biosynthesis in S. cerevisae are the same as those in E. coli; however, the first two steps may be different so that S. cerevisae cannot produce biotin from simple carbon sources. In addition, it was found recently that S. cerevisae possesses a BIO5 gene, which the gene product thereof is associated with the uptake of biotin and thus named as biotin permease (Phalip, V., et al., Gene 232:43–51 (1999)). With respect to edible yeast, Candida utilis, the biosynthesis of biotin remains unclear, and no related gene or enzyme has been identified. It is only understood that S. cerevisae should be cultured in the presence of biotin while C. utilis is not present, indicating that C. utilis is able to synthesize biotin from simple carbon sources.
Most organisms require biotin to survive, and only a few organisms can synthesize biotin themselves. The conditions due to lack of biotin in human and animals are not common; however, under some hereditary diseases or dystrophy, the lack of biotin usually results in severe consequence. For example, babies that lack holocarboxylase synthase usually have symptoms such as vomiting or asthma from the first day they are born; babies that lack biotindase may have disorders with skin, eye, and urinary system when 2–3 years old. In the past 10 years, it has been found that biotin is associated with the problems of farm animals that are bred on a large-scale. It is notable in poultry, for example, having fatty liver and kidney syndrome (FLKS), and acute death syndrome (ADS) in chicken. In 1977, it is also found that the cyllopodia (crooked-foot disease) in indoor-bred pigs is associated with the lack of biotin. Therefore, the demand of biotin increases 15% per year, in which 60–80% of biotin is used as feed additives.
In conventional methods, the production of biotin is carried out by chemical-synthetic processes, in which a crystalline biotin with high purity can be obtained for the use of medication. However, the separation and purification of the product requires very complicated steps. Moreover, in the prior art, methods for producing compounds by genetic engineering mostly employ the conventional replicative plasmid. It usually requires antibiotics to stabilize the copy number of the replicative plasmid, and thus, it is not appropriate for long-term fermentation and for food and feed. Therefore, there is still a need for development of yeast with high biotin-productivity for the use of feed additives, food additives, or cosmetics. Because the edible yeast itself is traditional food and feed, the yeast with high biotin-productivity prepared by the present invention can be formulated directly to merchandise after the processes of fermentation, separation, and drying without complicated purification. Further, the present invention uses integrated plasmid in lieu of replicative plasmid for the transformation of such genes into yeast genome, so that a stable yeast strain for high production of biotin is obtained.